BHD Exam IV

Clostridium and B. anthracis

Clostridia general natural history (6)

Gram positive, rod shaped (may be gram variable)

Endospore forming, often terminal/subterminal

Anaerobic fermentation, obligate anaerobes (no SOD/catalase)

Live in soil, organic matter, gut microbiota

Produce endotoxins, diseases include botulism, tetanus, colitis, and gangrene

Typically motile (other than C. perfringens), use peritrichous flagella

Note on identification of Clostridia (8)

Gram staining: +

Anaerobic culture: Clostridia are obligate anaerobes

Colony morphology: Black colonies on ChromID C. difficile agar

Spore formation

Hemolytic patterns: Observe hemolysins on blood agar; alpha hemolysis by C. perfringens alpha toxin

Biochemical tests: Phospholipase C activity for alpha toxin

Molecular diagnostics: PCR/Toxin assays to detect toxin genes or endotoxins directly

Gas production: Look for gas formation in liquid media, indicate anaerobic fermentation

Endospores: Specialized survival cells (4)

Dormant: Metabolically inactive, thick, dehydrated (heat-res.) cell wall

Highly resistant to heat, desiccation, staining, chemical sterilization, radiation

Autoclave invented to kill endospores

Sporulation → spore → Germination → Vegetative cell (cycle)

Major diseases caused by Clostridia and toxins (4)

C. botulinum: Botulism, Botulinum toxin (flaccid paralysis)

C. tetani: Tetanus, tetanospasmin (spasmic paralysis)

C. difficile: Antibiotic-related colitis, pseudomembranous colitis, TcdA, TcdB, CDT

C. perfringens: Food poisoning and gas gangrene, alpha toxin and enterotoxin

C. perfringens (7)

Non-motile, spread via toxins

Anaerobe, grows from 37-45C, large colonies on blood agar

Forms endospores

Virulence: Produces lots of toxin (alpha toxin, enterotoxin - CPE, PFO)

Causes gas gangrene, food poisoning, necrotizing enteritis, wound infections

Lives in soil, decaying matter, GI tracts

Food-borne transmission or wound infections (spore entry)

Alpha toxin (1)

Phospholipase C activity, hemolysis, tissue necrosis, and vascular damage

Alpha toxin mechanism (5)

Alpha toxin hydrolyzes SM and PC in host cell membranes

SM hydrolysis generates ceramide, which promotes apoptosis signaling

PLC activity produces DAG, activating PKC and NF-kB, leading to ROS and inflammatory cytokine (IL-8) production

Membrane disruption causes cell lysis

Alpha toxin promotes platelet aggregation, vasoconstriction, and endothelial damage

Enterotoxin (CPE) mechanism (4)

CPE binds to claudin proteins (tight junction components) on intestinal epithelial cells

After binding, the toxin oligomerizes to form pores in the plasma membrane

This leads to Ca2+ influx, activation of apoptosis pathways, and loss of epithelial integrity

Tight junction disruption increases intestinal permeability, causing fluid and electrolyte loss → diarrhea

PFO mechanism (4)

PFO is secreted as water-soluble monomers

They irreversibly attach themselves to target host cells by binding to cholesterol-rich microdomains

Once anchored, 35-50 monomers link together on membrane to form a pre-pore

The pre-pore undergoes a structural shift, causing the beta-sheet domains of PFO through lipid bilayer to form a massive, lethal pore in cell membrane

Clinical signs/syndromes: Food poisoning (7)

Enterotoxic infection: “food service germ”

Common cause of food poisoning (~1 million cases/year in U.S.)

Outbreaks usually linked to instituted with catered food

Commonly found on raw meat and poultry

Ingestion of spores (>10^8)

Coat protein causes toxic response

Water diarrhea, cramps, self-limiting, <24 hours

Clinical syndromes: Gas gangrene (6)

PFO and alpha toxin are the major contributors to pathology (not CPE)

Deep penetrating wound and introduction of spores: Lack of blood supply and lactic acid buildup

Bacteria germinate, multiply, invade: Tissue destruction, nutrient release

Usually polymicrobial, C. perfringens with Pseudomonas, E. coli, Streptococcus/Staphylococcus

Sugars broken down into formic acid → release of CO2/H2 gas

Usually amputation necessary, antibiotic treatment difficult

C. difficile (7)

Motile with peritrichous flagella, motile in liquid

Obligate anaerobe, grow best at 37C, yellow colonies on blood agar, black colonies on ChromID C. difficile agar

Forms endospores

Virulence factors include TcdA (enterotoxin), TcdB (cytotoxin), and CDT (binary toxin)

Diseases include antibiotic-associated diarrhea, pseudomembranous colitis, toxic megacolon, common nosocomial infection

Live in gut microbiota, persists as spores in environment such as hospitals

Transmitted by fecal-oral route, often by contaminated surfaces or hands

C. difficile toxins (4)

TcdA and TcdB have glycosyltransferase mechanism, turn off Rho GTPases

TcdA: Narrow host range, less potent (endotoxin), targets intestinal epithelial cells, causes fluid secretion

TcdB: Broader host range, more potent (cytotoxin), targets epithelial and immune cells, causes cytoskeletal disruption and death of cells

CDT: ADP-ribosylating toxin, modifies actin, disrupts cytoskeleton, enhances bacterial adhesion

TcdA/B mechanism (5)

TcdA can recognize a few different receptors on intestinal epithelium, causes clustering followed by clathrin-independent endocytosis

TcdB has much broader receptor range, causes clustering followed by clathrin-dependent endocytosis

Low pH in endosome causes transmembrane pore formation in toxins, APD and GTD translocated into cytosol

These toxins inactive Rho family GTPases by glycosylation, disrupting the actin cytoskeleton

Effects include tight junction collapse, cytokine production, cell rounding and apoptosis

CTD mechanism (5)

Recognizes lipoprotein receptor on gut epithelium

Clustering allows CDTa to bind, then toxin is endocytosed

Endosome acidifcation triggers CDTa translocation into cytosol

CDTa ADP-ribosylates actin, inhibiting actin polymerization, which promotes aberrant microtubule protrusion (supported by septins)

The protrusions can envelope C. difficile cells to augment adhesion to the host

C. difficile pathogenesis (6)

Disruption of normal flora (e.g. by broad spectrum antibiotics) →

Colonization of colon with C. difficile →

Toxin A and B production →

Diarrhea →

Colon ulceration →

Systemic disease, septic shock, and death

Understanding C. difficile (4)

Environmental survival: C. difficile produces spores, vegetative cells die outside the colon

Nosocomial risk: Found in 20% of hospitalized patients, often because of antibiotic therapy disrupting gut flora, allowing pathogen overgrowth

Symptoms: Diarrhea, pseudomembranous colitis - life threatening condition marked by inflammation and necrosis of colon

Treatment/relapse: Treated with vancomycin, through relapses are frequent due to spore survival and potential of reinfection

Epidemic strain (3)

Increased production of toxins A and B

Increased resistance to fluoroquinolones

Increased production of CDT (binary) toxin

Botulinum toxic (BoNT A-F; A most toxic) (3)

Inhibits neurotransmitter release, causing flaccid paralysis

Targets NMJ, cleaves SNARE proteins, blocking release of acetylcholine (which tells cells to contract)

Binds to synaptic vesicle proteins at the NMJ, internalized via endocytosis

Tetanus toxin/tetanospasmin (TeNT) (3)

Blocks inhibitory neurotransmitter release, causing spastic paralysis

Targets CNS inhibitory neurons, cleaves synaptobrevin, preventing GABA and glycine release (tells cells to relax)

Binds to peripheral nerve endings, transported by retrograde axonal transport to CNS

Clostridial neurotoxins (4)

Diseases are toxin-based, no bacteria are required once toxin is released

Specific binding to neuronal receptors

Cleavage of neuron-specific SNARE proteins

TeNT and BoNT (tetanus/tetanospasmin and botulinum toxins respectively)

Domains in botulinum toxin and tetanospasmin

Zinc endopeptidase - catalytic domain

Membrane translocation - translocation domain = HC N-term.

Binding domain - Receptor binding domain (RBD) = HC C-term.

Summary table: BoNT mechanism (6)

HCC domain binds synaptic vesicle proteins (SV2, synaptotagmin)

Endocytosis is mediated by the HCC domain

Acts locally at the NMJ

HCN forms a pore, allowing LC to enter cytosol

LC cleaves SNAP-25, VAMP, or syntaxin in excitatory neurons

Effect is blocking acetylcholine → Flaccid paralysis

Summary table: TeNT/tetanospasmin mechanism

HCC domain binds gangliosides on motor neurons

Endocytosis is mediated by the HCC domain

HCN domain facilitates retrograde axonal transport to CNS

HCN forms a pore, allowing LC to enter cytosol

LC cleaves VAMP (synaptobrevin) in inhibitory neurons

Effect is blocking GABA and glycine → Spastic paralysis

BoNT mechanism (10)

Binding to host receptors: RBD (HCC) binds synaptic vesicle proteins on cholinergic neurons at NMJ, ensures specificity

Endocytosis: RBD (HCC) mediates receptor-mediated endocytosis, forming an endosomal vesicle

Translocation: Translocation domain (HCN) forms a translocation pore under acidic endosomal conditions. Catalytic domain (LC) enters cytosol after reduction of disulfide bonds connecting the HC and LC. BoNT is secreted as an inactive protein, proteolytic cleavage by bacterial/host proteases activates BoNT

SNARE protein cleavage: Catalytic domain (LC) cleaves SNARE proteins (BoNT A/E: SNAP-25) (BoNTB/D/F/G: Synaptobrevin - VAMP) (BONT C: SNAP-25 and syntoxin)

Effect: Inhibits release of acetylcholine, leading to flaccid paralysis

TeNT/tetanospasmin mechanism (8)

Binding to host receptors: RBD (HCC) binds to gangliosides on motor neurons

Endocytosis: RBD (HCC) mediates receptor-mediated endocytosis, forming an endosomal vesicle

Retrograde transport: Translocation domain (HCN) facilitates retrograde axonal transport to inhibitory neurons in CNS

Translocation: Translocation domain (HCN) forms a pore in the acidic endosome. Catalytic domain (LC) enters the cytosol after the disulfide bond connecting it to HCN is reduced. Host proteases cleave the toxin into its active form

SNARE protein cleavage: Catalytic domain (LCC), a Zn-dependent endopeptidase, cleaves synaptobrevin in inhibitory neurons

Effect: Inhibits release of GABA and glycine, leading to spastic paralysis

Flaccid paralysis (2)

BoNT blocks acetylcholine release → muscle relaxation

Gains access via GI tract, transcytoses bloodstream, targets stimulatory muscle neurons in the PNS

Spastic paralysis (2)

TeNT blocks GABA and glycine release → muscle contraction

Works on CNS in infected wounds, delivered by retrograde axonal transport to CNS, targets inhibitory motor neurons in CNS

“Tightening” Illusion (3)

Botox does NOT physically shrink or pull the skin taut

The botulinum toxin blocks nerve signals, preventing muscle contraction

Since underlying muscle can’t contract and bunch the skin together, the skin can rest completely smooth and flat

C. botulinum (7)

Motile with peritrichous flagella for movement

Obligate anaerobe, 35C, 4.6-9 pH

Forms endospores

Virulence: Produces BoNT (A,B,E, and F cause disease in humans)

Diseases: Botulism (food-borne, wound, infant, iatrogenic); flaccid paralysis

Lives in soil, organic matter, spores are ubiquitous

Transmitted by food (toxin ingestion), wound (spore ingestion), infant (spore ingestion)

Infection with C. botulinum (7)

Two ways in which humans can be exposed to botulinum toxin:

• 1. Ingestion and inhalation of preformed toxin

• 2. Infection with C. botulinum spores and de novo synthesis of toxin by germinated organisms

-1. Entry: Adults: Toxin/wound infection; Infants: C. botulinum

-2. Absorption: Adults: Ingested toxin; Infants: Toxin made by C. botulinum infection

-3. Spread of toxin

-4. Disease: Flaccid paralysis, cardiac and respiratory failure

C. botulinum can cause infant/wound/food-borne botulism (3)

Infant (72%): Ingestion of viable spores, low mortality

Food-borne (25%): Caused by preformed botulinum toxin, spreads from GI tract to bloodstream

Wound (3%): When spores enter through an open wound, germinate, and produce toxin locally (like food-borne but no GI symptoms)

Prevention, Biothreat, Medicinal uses, and Treatment (5)

Prevention: Honey can contain spores, don’t feed to babies <12 months old, if home-canning, boil 10 mins before consumption

Biothreat agent: C. botulinum toxin is THE most potent bacterial toxin - category A biowarfare agent (33 million times more potent than cyanide!)

Medicinal uses of toxin: Therapeutic reagent against several neuromuscular disorders (migraines, wrinkles)

Treatment: Antitoxin with equine serum trivalent botulism antitoxin, neutralizes free-circulating toxin if promptly administered

• Botulism infections usually require 1-3 months of hospitalization!

C. tetani (7)

Motile with peritrichous flagella for movement

Obligate anaerobe, 37C

Produces terminal, drumstick-shaped endospores

Virulence: Produces tetanospasmin, a neurotoxin that targets inhibitory neurons → spastic paralysis

Diseases: Causes tetanus, characcterized by muscle stiffness, spasms, and potential respiratory failure

Lives in soil, dust, and animal intestines

Transmitted when spores enter wounds (especially deep oxygen deprived injuries/puncture wounds)

Pathogenesis of tetanus (9)

Entry and germination: Spore entry requires a low ID, typically enter via wounds; Germination conditions require low oxygen, 3-21 day incubation period

Toxin production: Toxin comprises 10% of cellular protein released from lysed bacteria! Systemically spread via lymphatics and blood, neural pathways (retrograde axonal transport)

Clinical manifestations: Early symptoms include spastic paralysis, lockjaw, stiff neck/abdomen; Severe symptoms include complete tetonic spasm, can be fatal if untreated

Clinical presentation of tetanus (2)

Generalized tetanus: 80% of cases, spasmic muscle contractions in one extremity or body region

Neonatal tetanus: Born to mothers without passive immunity

Treatment/prevention of tetanus (4)

Neutralize the unbound toxic by passive immunization

Wound management: Debridement to eradicate spores and necrotic tissue

Antimicrobial therapy: Penicillin

Immunize against tetanus, since natural disease does NOT confer immunity!